EP3682013B1 - Bacterial strain clostridium histolyticum and its use - Google Patents

Bacterial strain clostridium histolyticum and its use Download PDF

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EP3682013B1
EP3682013B1 EP18779553.9A EP18779553A EP3682013B1 EP 3682013 B1 EP3682013 B1 EP 3682013B1 EP 18779553 A EP18779553 A EP 18779553A EP 3682013 B1 EP3682013 B1 EP 3682013B1
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collagenase
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clostridium histolyticum
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Marek MOSA
Martin BENESIK
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Mb Pharma SRO
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    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
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Definitions

  • the invention is based on the new strain of bacterium Clostridium histolyticum, using it for production of crude collagenase with a high yield, collagenase prepared using this strain and application of the collagenase for isolation of Langerhans islets.
  • Collagenase is a protease which is able to degrade the collagen protein (the basic component of intercellular mass, e.g. in connective tissue). Collagenases are commonly used in research, particularly for disintegration of intercellular mass to release cells. In this respect, clostridiopeptidase A isolated from Clostridium histolyticum proved to be useful (the name of the bacterium, i.e. histolyticum, reflects its ability to degrade tissues). Enzyme collagenase is not only used for research purposes; it has found its place in human and veterinary medicine, especially for treatment of skin diseases.
  • the collagenase removes necrotic tissue from wounds and thus accelerates healing and improves epithelialization.
  • Collagenase is used in tissue transplantation and for tissue disintegration. It is also applied for treatment of (acid) burns of various degrees, decubiti, skin ulcers, scabs, etc. Not only the, epithelialization of skin is fast and effective after treatment with collagenase, the collagenase treatment also prevents formation of keloids (enlarged scars of tumour-like appearance) and hypertrophic growth as a result of formation of decomposed collagen.
  • Bacteria are cultured at 37 °C and pH 7.2.
  • Berman S., Lewenthal J.P., Webster M. E., Altieri P.L. and Gochenour R.B.: J. Bact. 82, 582 (1961 ) also examined growth conditions of Clostridium histolyticum with the aim to produce collagenase. They were successful in culturing of Clostridium histolyticum in medium which did not contain any inorganic salts; it only contained proteose peptone, enzymatically hydrolysed proteins of casein and soya (soya culture medium) and a vitamin solution.
  • Such a composition of culture medium also determined other parameters of conditions required for satisfactory growth and biosynthesis of collagenase, such as the value of pH 8.5 and a temperature 30 °C and higher.
  • Clostridium histolyticum is an anaerobic bacterium and anaerobic conditions must be ensured for culturing of this bacterium in liquid environment.
  • Takahashi S. and Seifert S.: J. Appl. Bact. 35, 47 (1972 ) used reducing agents sodium thioglycolate and sodium bisulfite in order to achieve anaerobic conditions necessary for bacterial growth.
  • the optimum results, i.e. the highest yield of collagenase was achieved when the above-mentioned reducing agents were used in the ratio 1:1.
  • the objective of this invention is to provide a specific Clostridium histolyticum strain by use of which the product of the collagenase enzyme is obtained and in its final form can be used for cleavage of connective structures of pancreas in order to receive vital Langerhans islets which can be used in a transplantation treatment of diabetes.
  • Collagenase is a mixture of up to 12 proteolytic enzymes and other proteins received from a filtrate of Clostridium histolyticum cultures. Pancreas digestion is performed in an isolation chamber which was designed by Dr. Ricordi et al.
  • the substance of the solution is bacterium Clostridium histolyticum CCM 8656 producing collagenase.
  • Collagenase produced by the strain has the following characteristics: It is a mixture of two collagenases coll and col 2 produced by the strain of bacterium Clostridium histolyticum (CCM 8656) with molecular weight of 116 kDa and 126 kDa.
  • the protein mixture contains collagenase and its natural degraded parts, which represent the most of the dry matter in the final product. On gel SDS PAGE both stripes must be apparent, and there is a possibility of occurrence of lower molecular weight fragments which still poses catalytic activity.
  • Both types of collagenase consist of two peptide domains which are able on their own to decompose collagen.
  • the rest of the protein consists of a binding domain which binds the whole enzyme to substrate, and even though it increases its activity via bonding to collagen it is not necessary for the activity. Smaller molecules of the catalytic domains thus complement activity of relatively large molecule of the complete enzymes thanks to better diffusion.
  • Collagenase must have specific activity higher than 700 PZS/g. This unit (PZS/g) is defined as a number of micromols of substrate degraded by one gram of enzyme within one minute at a temperature of 25 °C. Clostripain activity may be also desirable; however, it is not a must.
  • Clostripain may be the required admixture to the resulting collagenase mixture.
  • Unit U/mg is such enzyme activity of clostripain which catalyses hydrolysis of 1 ⁇ mol of substrate (BAEE) in 1 minute at a temperature of 25 oC, pH 7.6 and presence of 2.5 mmol/l DTT.
  • the optimum value of activity should range within 1.2-1.45 U/mg. It is therefore a naturally produced mixture of collagenolytic enzymes prepared by partial purification (see examples) and this mixture is suitable for isolation of LI.
  • the strain of bacterium Clostridium histolyticum with working identification MB204 is deposited in the CCM - Czech Collection of Microorganisms, Kamenice 5, 625 00 Brno of 20/01/2016 under No. CCM 8656.
  • the strain Clostridium histolyticum CCM 8656 which we have cultivated (via selection by long-term passaging) was obtained from a mixture of 3 strains that were obtained from a German collection of strains as strains DSM 627, 1126 and 2158.
  • Culturing is generally performed in tryptone medium, suitable for sufficient production of collagenase: (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na2HPO4 3.4 g/l) pH 8.4, before inoculation, 100 ⁇ l of vitamin K of storage concentration 1% and 100 ⁇ l of L-cysteine of storage concentration 0.5 g/ml is added to the culturing medium. The culture is cultivated 24 hours at 37 °C ⁇ 1 °C without agitation. In the strain, using controlled evolution and long-term passaging (adaptive laboratory evolution) ( Dragonits, 2013 . Dragosits M, Mattanovich D.
  • composition of the tryptone medium suitable for sufficient production of collagenase with collagen (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na2HPO4 3.4 g/l, collagen 50 g/l) pH 8.4, before inoculation, 100 ⁇ l of vitamin K of storage concentration 1% and 100 ⁇ l L-of cysteine of storage concentration 0.5 g/ml are added into the cultivation medium.
  • the culture is cultivated 72 hours at 37 °C ⁇ 1 °C without agitation, ideally in anaerobic conditions. Genes encoding collagenase ColG and ColH were identified previously in C.
  • Clostridium histolyticum is an anaerobic gram-positive bacterium. Bacterial cells are mobile peritrichal straight rods with a size of 0.5-0.9 ⁇ 1.3-9.2 ⁇ m and they form in pairs or short chains. Cells are capable of sporulation; in anaerobic conditions their cell wall contains meso-DAP, glutamic acid and alanine.
  • the strains of this bacterium are sensitive to chloramphenicol, erythromycine and tetracycline.
  • the strains are toxic but in long cultivation their precursor-proteases may degrade own toxins.
  • Specific 16S RNA GenBank accession number 16S rRNA of gene: M590944 shows that this strain is similar to the strains Clostridium limosum (97.2%) and Clostridium proteolyticum (96.1%).
  • Collagenase for isolation of Langerhans islets is received as a metabolite of the bacterium Clostridium histolyticum. This enzyme has a synergic effect in the process of degradation of collagen and other extracellular components. Collagenase for isolation of Langerhans islets is generally appropriate for isolation of cells from a number of animal tissues. The product is provided with basic information about enzymatic activity. The optimum concentration of enzyme and the specific conditions for using of degradation of various tissues has to be defined empirically. The enzyme is not of animal origin.
  • Collagenase for isolation of Langerhans islets is suitable for cell separations of e.g. tumour cells, separation of murine kidney cells, cells of lung tissue and various epithelial tissues.
  • the enzyme can be also used for isolation of hepatocytes. Considering the selective collagenolytic activity which primarily does not damage cell membranes, the enzyme can be used as generally dispersing cell agent, also in the conditions of cell cultures. It is generally applicable that organs with a higher content of collagen can be incubated with collagenase for isolation of Langerhans islets for a longer time and at concentrations higher than when working with other proteolytic enzymes, without the cells losing their viability.
  • Bacterial culture Clostridium histolyticum, strain No. CCM 8656 is stored in a freezing medium at -80 °C or in a form of lyophilizate in a glass vial at 4 °C.
  • 100 ⁇ l of de-frost culture is inoculated to 100 ml of a liquid medium which is sterilized immediately before inoculation and then temperature-adjusted to a temperature 37°C.
  • the culture medium, suitable for cultivation of anaerobic bacteria is thus deaerated.
  • composition of tryptone medium suitable for sufficient production of collagenase (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na 2 HPO 4 3.4 g/l) pH 7.8, before inoculation, 100 ⁇ l of vitamin K of storage concentration 1 % and 100 ⁇ l of L-cysteine of storage concentration 0.5 g/ml are added to the culturing medium. The culture is cultivated 24 hours at 37 °C ⁇ 1 °C without agitation. After cultivation purity of bacterial strains is verified through growth on solid media (anaerobic cultivation) and morphology of cells is assessed microscopically.
  • Bacterial culture Clostridium histolyticum, strain No. 8656 cultivated in 100 ml of medium is collected from the bottom part of a bottle. 5 ml of culture is inoculated to 500 ml of a liquid medium which is sterilized immediately before inoculation and then temperature-adjusted to a temperature 37 °C. The culture medium, suitable for cultivation of anaerobic bacteria is thus deaerated.
  • composition of tryptone medium suitable for sufficient production of collagenase: (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na 2 HPO 4 3.4 g/l) pH 7.8, before inoculation, 100 ⁇ l of vitamin K of storage concentration 1 % and 100 ⁇ l of L-cysteine of storage concentration 0.5 g/ml are added to the culturing medium. The culture is cultivated 24 hours at 37 °C ⁇ 1 °C without agitation.
  • Cultivation in Fermentor 19 litres of cultivation medium is prepared in a fermentor (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Ha 2 HPO 4 3.4 g/l) pH 7.8 before inoculation 20 ml of vitamin K (storage concentration 1 %) and 20 ml of L-cysteine (storage concentration 0.5 g/ml) are added. Fermentor is inoculated with 500 ml of bacterial culture from the previous step of cultivation. Mixed inoculum is sucked aseptically to the prepared cultivation medium. After inoculation sample 0 is taken. Culturing proceeds at +37 °C ⁇ 1°C. Culturing conditions are adjusted depending on bacterial growth. Since cultivation starts, pH is adjusted on a continuous basis with a 2M solution of NaOH to value of 7.8 ⁇ 0.5. Culturing proceeds without stirring or aeration!
  • Cultivation is finished if 2 consecutive samplings do not show a significant increment and pH does not change. If at least pH changes, cultivation lasts for 40 - 48 hours.
  • the dialysing hose is placed in 10 I of buffer Tris Cl (Tris 0.75 g/l, CaCl 2 0.484 g/l pH 10) and left to dialyse for 3 hours. Then the buffer is replaced with other 10 l of TRIS HCI for 24 hours. Then the buffer is replaced with 10 l of TRIS HCl of a different pH (Tris 0.36 g/l, CaCl 2 0.242 g/l pH 7.5 - 8.5) for 24 hours. Then it is again replaced with fresh Tris III. If dialysate appears to be to thick, replacement of the Tris buffer may be done two more times within further 24 hours.
  • Tris Cl Tris 0.75 g/l, CaCl 2 0.484 g/l pH 10
  • Clostridium histolyticum CCM 8656 is the same as in Example 1.
  • bacteria C. histolyticum is removed by centrifugation (7000 ⁇ g) and precipitation of collagenase with ammonium sulphate is performed only in supernatant.
  • Precipitate containing sulphate with precipitated protein is left to sediment for 4 - 6 days at 4 °C.
  • Tris HCl buffers (the same buffers as in Example 1), then ultrafiltration follows again in cartridges millipore with "cut off" 50 kDa.
  • the sample is filled by 10 ml in vials and lyophilised.
  • Clostridium histolyticum CCM 8656 is the same as in Example 1. However, dialysis is followed by ultrafiltration through ultrafiltration cartridges, at first with "cut-off" 300 kDa for removal of ballast proteins of a large molecular weight. Thus volume is increased because flow-through is maintained and then the solution is ultrafiltrated in a cartridge "cut off” 50 kDa for removal of smaller proteins and degraded parts.
  • Tris-HCI buffer Tris 0.36 g/l, CaCl 2 0.242 g/l pH 7.5 - 8.5
  • the sample is filled by 10 ml in vials and lyophilised.
  • Clostridium histolyticum CCM 8656 Inoculation and culturing of bacterial strain Clostridium histolyticum CCM 8656 is the same as in Example 1.
  • bacteria Clostridium histolyticum are removed by centrifugation (7 000 ⁇ g).
  • precipitation with ammonium sulphate is not used; protein is directly thickened.
  • Trough ultrafiltration cartridges at first with "cut off" 300 kDa ballast proteins of a large molecular weight are removed. Thus volume is increased because flow-through is maintained and then the solution is ultrafiltered in a cartridge with "cut off” 50 kDa for removal of smaller proteins and degraded parts, and volume is reduced.
  • the sample is filled by 10 ml in vials and lyophilised.
  • the method uses only precipitation with ammonium sulphate, dialysis and an ultrafiltration system, which is a simple, easy-to-do method, compared to the costly and demanding purification method using chromatography. Moreover, large collagenase volumes can be processed using this method, compared to chromatographic methods. This method preserves other proteins which in a proper ratio support collagenase activity. Since bacterial culture is processed in the production, the intracellular proteins get to the final product. In other cases, only supernatant after culturing is processed, and here the supporting enzymes are lost.
  • the method is also innovative in using two pH values, at first precipitate is dialysed against a buffer of a high pH. This process has been implemented in order to increase protein stability and achieve a higher yield.
  • Dendo et al. (2015) demonstrates that the synergy of collagenase and clostripain positively affects the amount of LI obtained from pancreases of experimental animals.
  • Combination of enzymes causes high yield >1000 LI, similarly as in case of our crude collagenase (Table 1 ).
  • the publication deals with highly purified recombinant proteins.
  • the collagenase mentioned in the publication is not produced directly from C. histolyticum, it is a GMO product - a recombinant protein produced in E. coli. Adjusting to the desired concentration is performed separately for each enzyme.
  • the mixture of enzymes comes directly from specific strain C. histolyticum CCM 8656 and the quality of the mixture, showed by the amount of isolated LI, is comparable to recombinant collagenase.
  • Clostridium histolyticum which produce not only bacterial collagenase but a significant amount of clostripain and neutral proteases. These enzymes may, but may not be required for production of the final product. In the case they are absent, they must be then added separately in order to isolate the Langerhans islets. Clostripain and neutral proteases are present in the mixture of proteins produced by C. histolyticum CCM 8656 using the methods mentioned above.
  • Working identification numbers of the three strains are: MB 201, MB 202 and MB 204 (CCM 8656), and for the purpose of the mass production the strain Clostridium histolyticum MB 204 (CCM 8656) was selected.
  • CCM 8656 the gene for collagenase in this production strain was confirmed using PCR, for better characterization it is better to determine the whole sequence of genome DNA. Thus, the genes for clostripain and potential neutral proteases can be found. Therefore, to ensure successful sequencing, DNA of very good quality and concentration must be prepared. A large volume of data must be processed for sequencing, and finished sequences of contigs must be then annotated. All three strains of C.
  • strain M 201 contained a gene for col 1 in two copies an gene for col 2 in one copy only; on the contrary, strain MB 202 has gene col 1 in one copy and gene col 2 in two copies.
  • the production strain MB 204 had both genes col 1 and col 2 in one copy only.
  • Genomes of all strains include a gene for clostripain. This enzyme can help collagenase to better disintegrate tissues during isolation of Langerhans islets.
  • the main component of the collagenase final mixture should be collagenase, while clostripain should be a minor component. This was demonstrated.
  • Clostridium histolyticum dispose of gene for production of clostripain but alternatively, it would be more appropriate for the proper ratio of collagenase and clostripain to produce both enzymes separately and produce a mixture of the proper ratio just before the application.
  • Clostripain has molecular weight 43.4 kDa so it would be possible to produce it relatively easily also as a recombinant protein e.g. in E. coli.
  • Our strain CCM 8656 produces clostripain in an optimal amount to support the efficacy of collagenase in LI isolation.
  • collagenase and its natural degraded parts represent the most of the dry matter in the final product. It may also contain clostripain or neutral proteases detectable using an appropriate method for measuring of activity of those admixtures. However, presence of both non-degraded collagenases and possibly their parts is essential. There must be two stripes visible on gel 12% SDS-PAGE, corresponding to the size 116 kDa and 126 kDa, and there is also a possibility of occurrence of stripe of lower molecular weight protein, which still has catalytic activity. If zymogram is used, lytic zones of proteins of lower molecular weight may be visible.
  • Collagenase must have specific activity higher than 700 PZS/g. This unit is defined as a number of micromols of substrate degraded by one gram of enzyme within one minute at a temperature of 25 °C. Clostripain activity may be also desirable; however, it is not a must. Clostripain may be the required admixture to the resulting collagenase mixture. Unit U/mg is such enzyme activity of clostripain which catalyses hydrolysis of 1 ⁇ mol of substrate (BAEE) in 1 minute at a temperature of 25 oC, pH 7.6 and presence of 2.5 mmol/l DTT. The optimum value of activity should range within 1.2-1.45 U/mg.
  • the gene encoding collagenase in MB 204 has the same nucleotide sequence as the analogous gene in the strain 2158, but the strain MB 204 produces more efficient mixture of enzymes, thus a markedly different phenotypic manifestation is observed at equal cultivation conditions. Even though the genes encoding collagenase are identical in these two strains, the expression of the protein in the cell can be affected by various regulation mechanisms, in which the two strains differ. Although compared to commercially used bacteria this strain contains one copy of the gene for collagenase type I and one copy of the gene for collagenase type II; however, the yield of collagenase increased, most probably thanks to expression caused by a strong promotor. Moreover, the strain produces a minimal amount of toxic proteins, and during preparation undesirable proteins are degraded which is a big advantage for practical use in transplantation medicine.
  • the new method of production in combination with the new strain leads to production of stable collagenase from Clostridium histolyticum. Cultivation of relatively small volume gives sufficient yields of both types of collagenase in the final product. Although partial degradation occurs, it is more likely beneficial in combination with sufficient amount of non-degraded part of the enzyme, as the degraded parts still retain their proteolytic activity.
  • Enzyme collagenase is used for digestion of pancreatic tissue, during which Langerhans islets (LI) are released and can be used for treatment of diabetes. Isolated LI using collagenase must meet strict criteria so they can be transplanted to diabetic patients. The most important monitored parameters of isolation are as follows: duration of digestion, digestion quality, quality of separation of cells from exocrine tissue and quality of cells after isolation and culturing. Quality of cells is evaluated using dying of viable and dead cells, using glucose-stimulated secretion of insulin with beta cells of LI (expressed as stimulation index) and using measuring of oxygen consumption. Using collagenase according to the invention resulted in achieving the digestion time of 15 min and average yield 1000 LI which retain sufficient vitality (more than 95 %). This collagenase is thus suitable for isolation of LI for transplantation purposes.

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CZ2017-537A CZ308157B6 (cs) 2017-09-13 2017-09-13 Kmen bakterie Clostridium histolyticum, kolagenáza připravená s použitím tohoto kmene a její použití
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CN113215058B (zh) * 2021-06-08 2022-05-03 山东大学 一株能够降解胶原蛋白的菌株及其应用
CZ309541B6 (cs) 2021-08-30 2023-03-29 MB PHARMA s.r.o. Kmen produkující clostripain-like protein pro použití ve směsi s rekombinantní kolagenázou ColG a ColH pro izolaci Langerhansových ostrůvků pankreatu

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JP5698536B2 (ja) * 2008-11-19 2015-04-08 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 アフィニティタグが結合した融合コラゲナーゼおよびその製造法
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